Resolution of transcription-induced hexasome-nucleosome complexes by Chd1 and FACT.

To maintain the nucleosome organization of transcribed genes, ATP-dependent chromatin remodelers collaborate with histone chaperones. Here, we show that at the 5' ends of yeast genes, RNA polymerase II (RNAPII) generates hexasomes that occur directly adjacent to nucleosomes. The resulting hexasome-nucleosome complexes are then resolved by Chd1. We present two cryoelectron microscopy (cryo-EM) structures of Chd1 bound to a hexasome-nucleosome complex before and after restoration of the missing inner H2A/H2B dimer by FACT. Chd1 uniquely interacts with the complex, positioning its ATPase domain to shift the hexasome away from the nucleosome. In the absence of the inner H2A/H2B dimer, its DNA-binding domain (DBD) packs against the ATPase domain, suggesting an inhibited state. Restoration of the dimer by FACT triggers a rearrangement that displaces the DBD and stimulates Chd1 remodeling. Our results demonstrate how chromatin remodelers interact with a complex nucleosome assembly and suggest how Chd1 and FACT jointly support transcription by RNAPII.

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner.

The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails, and binding partially disassembled nucleosomes. Here, we systematically test these and other scenarios in Saccharomyces cerevisiae and find that FACT binds transcribed chromatin, not RNAPII. Through a combination of high-resolution genome-wide mapping, single-molecule tracking, and mathematical modeling, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.

Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression.

Aberrant expression of programmed death ligand-1 (PD-L1) in tumor cells promotes cancer progression by suppressing cancer immunity. The retinoblastoma protein RB is a tumor suppressor known to regulate the cell cycle, DNA damage response, and differentiation. Here, we demonstrate that RB interacts with nuclear factor κB (NF-κB) protein p65 and that their interaction is primarily dependent on CDK4/6-mediated serine-249/threonine-252 (S249/T252) phosphorylation of RB. RNA-seq analysis shows a subset of NF-κB pathway genes including PD-L1 are selectively upregulated by RB knockdown or CDK4/6 inhibitor. S249/T252-phosphorylated RB inversely correlates with PD-L1 expression in patient samples. Expression of a RB-derived S249/T252 phosphorylation-mimetic peptide suppresses radiotherapy-induced upregulation of PD-L1 and augments therapeutic efficacy of radiation in vivo. Our findings reveal a previously unrecognized tumor suppressor function of hyperphosphorylated RB in suppressing NF-κB activity and PD-L1 expression and suggest that the RB-NF-κB axis can be exploited to overcome cancer immune evasion triggered by conventional or targeted therapies.

The Chd1 Chromatin Remodeler Shifts Nucleosomal DNA Bidirectionally as a Monomer.

Chromatin remodelers catalyze dynamic packaging of the genome by carrying out nucleosome assembly/disassembly, histone exchange, and nucleosome repositioning. Remodeling results in evenly spaced nucleosomes, which requires probing both sides of the nucleosome, yet the way remodelers organize sliding activity to achieve this task is not understood. Here, we show that the monomeric Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome. During sliding, Chd1 generates unstable remodeling intermediates that spontaneously relax to a pre-remodeled position. We demonstrate that nucleosome sliding is tightly controlled by two regulatory domains: the DNA-binding domain, which interferes with sliding when its range is limited by a truncated linking segment, and the chromodomains, which play a key role in substrate discrimination. We propose that active interplay of the ATPase motor with the regulatory domains may promote dynamic nucleosome structures uniquely suited for histone exchange and chromatin reorganization during transcription.

Identification and functional characteristics of CHD1L gene variants implicated in human Müllerian duct anomalies.

BACKGROUND: Müllerian duct anomalies (MDAs) are congenital developmental disorders that present as a series of abnormalities within the reproductive tracts of females. Genetic factors are linked to MDAs and recent advancements in whole-exome sequencing (WES) provide innovative perspectives in this field. However, relevant mechanism has only been investigated in a restricted manner without clear elucidation of respective observations. METHODS: Our previous study reported that 2 of 12 patients with MDAs harbored the CHD1L variant c.348-1G>C. Subsequently, an additional 85 MDAs patients were recruited. Variants in CHD1L were screened through the in-house database of WES performed in the cohort and two cases were identified. One presented with partial septate uterus with left renal agenesis and the other with complete septate uterus, duplicated cervices and longitudinal vaginal septum. The pathogenicity of the discovered variants was further assessed by molecular dynamics simulation and various functional assays. RESULTS: Ultimately, two novel heterozygous CHD1L variants, including a missense variant c.956G>A (p.R319Q) and a nonsense variant c.1831C>T (p.R611*) were observed. The variants were absent in 100 controls. Altogether, the contribution yield of CHD1L to MDAs was calculated as 4.12% (4/97). All three variants were assessed as pathogenic through various functional analysis. The splice-site variant c.348-1G>C resulted in a 11 bp sequence skipping in exon 4 of CHD1L and led to nonsense mediated decay of its transcripts. Unlike WT CHD1L, the truncated R611* protein mislocalized to the cytoplasm, abolish the ability of CHD1L to promote cell migration and failed to interact with PARP1 owing to the loss of macro domain. The R319Q variant exhibited conformational disparities and showed abnormal protein recruitment behavior through laser microirradiation comparing with the WT CHD1L. All these variants impaired the CHD1L function in DNA damage repair, thus participating in MDAs. CONCLUSIONS: The current study not only expands the mutational spectrum of CHD1L in MDAs but determines three variants as pathogenic according to ACMG guidelines with reliable functional evidence. Additionally, the impairment in DNA damage repair is an underlying mechanism involved in MDAs.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling.

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD+ A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

CHD1L Regulates Cell Survival in Breast Cancer and Its Inhibition by OTI-611 Impedes the DNA Damage Response and Induces PARthanatos.

The Chromodomain helicase DNA-binding protein 1-like (CHD1L) is a nucleosome remodeling enzyme, which plays a key role in chromatin relaxation during the DNA damage response. Genome editing has shown that deletion of CHD1L sensitizes cells to PARPi, but the effect of its pharmacological inhibition has not been defined. Triple-negative breast cancer SUM149PT, HCC1937, and MDA-MB-231 cells were used to assess the mechanism of action of the CHD1Li OTI-611. Cytotoxicity as a single agent or in combination with standard-of-care treatments was assessed in tumor organoids. Immunofluorescence was used to assess the translocation of PAR and AIF to the cytoplasm or the nucleus and to study markers of DNA damage or apoptosis. Trapping of PARP1/2 or CHD1L onto chromatin was also assessed by in situ subcellular fractionation and immunofluorescence and validated by Western blot. We show that the inhibition of CHD1L's ATPase activity by OTI-611 is cytotoxic to triple-negative breast cancer tumor organoids and synergizes with PARPi and chemotherapy independently of the BRCA mutation status. The inhibition of the remodeling function blocks the phosphorylation of H2AX, traps CHD1L on chromatin, and leaves PAR chains on PARP1/2 open for hydrolysis. PAR hydrolysis traps PARP1/2 at DNA damage sites and mediates PAR translocation to the cytoplasm, release of AIF from the mitochondria, and induction of PARthanatos. The targeted inhibition of CHD1L's oncogenic function by OTI-611 signifies an innovative therapeutic strategy for breast cancer and other cancers. This approach capitalizes on CHD1L-mediated DNA repair and cell survival vulnerabilities, thereby creating synergy with standard-of-care therapies.

Sex identification of birds in Taipei Zoo.

As a conservation and breeding institution for birds, Taipei Zoo plays an important role in restoring endangered species. As approximately half of all bird species are monomorphic, precisely confirming the sex of individuals is critical for the management of ex-situ conservation breeding populations, as well as for understanding the sex ratio of those in the wild. Generally, PCR is used more reliably for sex determination versus traditional methods such as plumage, behavior or hormone levels. Nevertheless, the various primer sets and annealing temperatures vary between species, and so inaccurate sexing can occasionally happen due to inadequate PCR conditions. To reduce the probability of misidentification, and to establish a PCR condition database for sex determination across the diverse range of avian taxa, we tested multiple primer sets and annealing temperatures for amplification of the bird sex-specific gene fragments (CHD1) for each captive or rescued avian species held at Taipei Zoo since 2014. A total of 162 species across 22 orders were tested using one or two primer sets. One hundred and fifty-five species were successfully sexed by the primer set 2550F/2718R and the success rate of sex typing reached over 90% of species tested in each order. Most species have suitable PCR annealing temperatures between 45°C and 55°C, and the species in the same avian taxa showed similar results in temperature. This indicates that it is possible to select the annealing temperature of other species in the same family when the species had not been tested before. We expect this study will improve the success rate of identifying sex by using applicable PCR conditions and reduce the time for searching references every time before attempts to PCR sex birds.

Coregulators Reside within Drosophila Ecdysone-Inducible Loci before and after Ecdysone Treatment.

Ecdysone signaling in Drosophila remains a popular model for investigating the mechanisms of steroid action in eukaryotes. The ecdysone receptor EcR can effectively bind ecdysone-response elements with or without the presence of a hormone. For years, EcR enhancers were thought to respond to ecdysone via recruiting coactivator complexes, which replace corepressors and stimulate transcription. However, the exact mechanism of transcription activation by ecdysone remains unclear. Here, we present experimental data on 11 various coregulators at ecdysone-responsive loci of Drosophila S2 cells. We describe the regulatory elements where coregulators reside within these loci and assess changes in their binding levels following 20-hydroxyecdysone treatment. In the current study, we detected the presence of some coregulators at the TSSs (active and inactive) and boundaries marked with CP190 rather than enhancers of the ecdysone-responsive loci where EcR binds. We observed minor changes in the coregulators' binding level. Most were present at inducible loci before and after 20-hydroxyecdysone treatment. Our findings suggest that: (1) coregulators can activate a particular TSS operating from some distal region (which could be an enhancer, boundary regulatory region, or inactive TSS); (2) coregulators are not recruited after 20-hydroxyecdysone treatment to the responsive loci; rather, their functional activity changes (shown as an increase in H3K27 acetylation marks generated by CBP/p300/Nejire acetyltransferase). Taken together, our findings imply that the 20-hydroxyecdysone signal enhances the functional activity of coregulators rather than promoting their binding to regulatory regions during the ecdysone response.

Using an aquatic model, Xenopus laevis, to uncover the role of chromodomain 1 in craniofacial disorders.

The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of CHD1 can result in intellectual disabilities and craniofacial malformations in human patients including cleft palate and Pilarowski-Bjornsson Syndrome. We have used the aquatic developmental model organism Xenopus laevis, to determine a specific role for Chd1 in such cranioafcial disorders. Protein and gene knockdown techniques in Xenopus, including antisense oligos and mosaic Crispr/Cas9-mediated mutagenesis, recapitulated the craniofacial defects observed in humans. Further analysis indicated that embryos deficient in Chd1 had defects in cranial neural crest development and jaw cartilage morphology. Additionally, flow cytometry and immunohistochemistry revealed that decreased Chd1 resulted in increased in apoptosis in the developing head. Together, these experiments demonstrate that Chd1 is critical for fundamental processes and cell survival in craniofacial development. We also presented evidence that Chd1 is regulated by retinoic acid signaling during craniofacial development. Expression levels of chd1 mRNA, specifically in the head, were increased by RAR agonist exposure and decreased upon antagonist treatment. Subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergized to result in orofacial defects. Further, RAR DNA binding sequences (RAREs) were detected in chd1 regulatory regions by bioinformatic analysis. In summary, by combining human genetics and experiments in an aquatic model we now have a better understanding of the role of CHD1 in craniofacial disorders.

SPOP and CHD1 alterations in prostate cancer: Relationship with PTEN loss, tumor grade, perineural infiltration, and PSA recurrence.

BACKGROUND: In the non-ETS fusion of prostate cancer (PCa) pathway, SPOP mutations emerge as a distinct oncogenic driver subclass. Both SPOP downregulation and mutation can lead to SPOP target stabilization promoting dysregulation of key regulatory pathways. CHD1 gene is commonly deleted in PCa. CHD1 loss significantly co-occurs with SPOP mutations, resulting in a PCa subclass with increased AR transcriptional activity and with a specific epigenetic pattern. METHODS: In this study, SPOP alterations at mutational and protein levels and CHD1 copy number alterations have been analyzed and correlated with ERG and PTEN protein expression and with the clinical pathological features of the patients. RESULTS: SPOP protein loss has been detected in 42.9% of the cases, and it has been strongly associated with PTEN protein loss (p < .001). CHD1 gene loss has been detected in 24.5% and SPOP mutations in 5.9% of the cases. Loss of CHD1 has been strongly associated with SPOP mutations (p = .003) and has shown a trend to be associated with ERG wt cancers (p = .08). The loss of SPOP protein (p = .01) and the combination of PTEN and SPOP protein loss (p = .002) were both statistically more common in grade group 5 cancers, with a prevalence of 60% and 37.5%, respectively. Furthermore, SPOP loss/PTEN loss and SPOP wt/PTEN loss phenotypes were strongly associated with extraprostatic perineural infiltration (p = .007). Strong CHD1 loss was associated with a shorter time to PSA recurrence in the univariate (p = .04), and showed a trend to be associated with the PSA recurrence risk in the multivariate analysis (p = .058). CONCLUSIONS: The results of the present study suggest that the loss of SPOP protein expression, either alone or in combination with loss of PTEN and, on the other hand, a marked loss of the CHD1 gene are very promising prognostic biomarkers in PCa.

CHD1 and SPOP synergistically protect prostate epithelial cells from DNA damage.

BACKGROUND: Recent genomic profiling has identified a subtype of prostate cancer (PCa) characterized by two key genetic alterations: missense mutation of speckle-type POZ protein (SPOP) and homozygous deletion of chromodomain helicase DNA-binding protein 1 (CHD1). Mutually exclusive with E26 transformation-specific (ETS) rearrangements, this subtype displays high genomic instability. Previous studies indicate that deficient SPOP or CHD1 alone leads to feeble prostate abnormalities and each protein is involved in DNA damage response (DDR). It remains to be determined whether CHD1 and SPOP cooperate to suppress prostate tumorigenesis and DDR. METHODS: Prostate-specific single or double knockout of Spop and Chd1 was generated with the Cre/loxP system in mice. Wild-type or mutant SPOP (F102C, F133V) overexpression and CHD1 knockdown with short hairpin RNA were created in human benign prostatic hyperplasia cell line BPH1. The levels of DNA damage and homologous recombination repair were measured by immunofluorescence staining of γH2AX and RAD51, respectively. RESULTS: Spop/Chd1 double-knockout mice displayed prostatic intraepithelial neoplasia at both young (3 months) and old (12 months) ages and failed to generate prostate adenocarcinoma. Compared with wild-type or single-knockout mice, the double-knockout prostate harbored moderately higher proliferating cells and dramatically augmented the level of γH2AX staining, although androgen receptor-positive cells and apoptotic cells remained at a similar level. In BPH1 cell line, SPOP mutant overexpression and CHD1 silencing synergistically sensitized the cells to DNA damage by camptothecin, an inducer of double-strand breaks. CONCLUSIONS: Our results indicate that SPOP and CHD1 can synergistically promote repair of naturally occurring or chemically induced DNA damages in prostate epithelial cells. Regarding the progression of the SPOP/CHD1 subtype of PCa, other functionally complementary drivers warrant further identification. The clinical implication is that this subtype of PCa may be particularly sensitive to poly(ADP-ribose) polymerase inhibitors or DNA-damaging agents.

Suppressing CHD1L reduces the proliferation and chemoresistance in osteosarcoma.

Osteosarcoma (OS) is the most common bone malignant tumor. However, the genetic basis of OS pathogenesis is still not understood, and occurrence of chemo-resistance is a major reason for the high morbidity of OS patients. Recently, chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) has been identified as a gene related to malignant tumor progression. Unfortunately, its effects on OS development and drug resistance are still not understood. In the study, we attempted to investigate the effects of CHD1L on tumorigenesis and chemoresistance in OS. We found that CHD1L expression was markedly up-regulated in OS samples, especially in cisplatin (cDDP)-resistant patients. We also showed that OS cells with CHD1L knockdown were more sensitive to cDDP treatment with lower IC50 values. In addition, we found that CHD1L deletion markedly reduced cell proliferation and induced apoptosis in OS cells with cDDP resistance. Moreover, the properties of cancer stem cells were highly suppressed in cDDP-resistant OS cells following CHD1L knockdown. Furthermore, multidrug resistance protein 1 (MDR-1) expression levels were dramatically decreased in OS cells with cDDP resistance when CHD1L was suppressed. Functional analysis indicated that CHD1L knockdown clearly restrained the activation of ERK1/2, protein kinase B (AKT) and NF-κB signaling pathways in cDDP-resistant OS cells. Consistently, animal experiments suggested that CHD1L suppression mitigated cDDP resistance in the generated in vivo xenografts. Collectively, CHD1L could modulate chemoresistance of OS cells to cDDP, and thus may be inspiring findings for overcoming drug resistance in OS.

The Chd1 chromatin remodeler forms long-lived complexes with nucleosomes in the presence of ADP·BeF3- and transition state analogs.

Chromatin remodelers use helicase-like ATPase domains to reorganize histone-DNA contacts within the nucleosome. Like other remodelers, the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler repositions nucleosomes by altering DNA topology at its internal binding site on the nucleosome, coupling different degrees of DNA twist and DNA movement to distinct nucleotide-bound states of the ATPase motor. In this work, we used a competition assay to study how variations in the bound nucleotide, Chd1, and the nucleosome substrate affect stability of Chd1-nucleosome complexes. We found that Chd1-nucleosome complexes formed in nucleotide-free or ADP conditions were relatively unstable and dissociated within 30 s, whereas those with the nonhydrolyzable ATP analog AMP-PNP had a mean lifetime of 4.8 ± 0.7 min. Chd1-nucleosome complexes were remarkably stable with ADP·BeF3- and the transition state analogs ADP·AlFX and ADP·MgFX, being resistant to competitor nucleosome over a 24-h period. For the tight ADP·BeF3--stabilized complex, Mg2+ was a critical component that did not freely exchange, and formation of these long-lived complexes had a slow, concentration-dependent step. The ADP·BeF3--stabilized complex did not require the Chd1 DNA-binding domain nor the histone H4 tail and appeared relatively insensitive to sequence differences on either side of the Widom 601 sequence. Interestingly, the complex remained stable in ADP·BeF3- even when nucleosomes contained single-stranded gaps that disrupted most DNA contacts with the guide strand. This finding suggests that binding via the tracking strand alone is sufficient for stabilizing the complex in a hydrolysis-competent state.

Organ-specific regulation of CHD1 by acute PTEN and p53 loss in mice.

Homozygous deletion of chromodomain helicase DNA binding protein 1 (CHD1) is among the most frequent genetic alterations in prostate cancer. CHD1 is converted from a non-essential to an essential gene for prostate cancer cell survival when phosphatase and tensin homolog (PTEN), another frequently deleted gene in prostate cancer, is disrupted. It remains unknown whether this PTEN-CHD1 genetic and functional relationship also operates in other solid tumors. Here, we address this question by using genetically engineered mouse models. Inducible deletion of Pten and p53 in all somatic cells of adult mice led to widespread PI3K/Akt pathway activation and hyperplastic phenotypes, causing multi-organ failure and lethality. Remarkably, when Chd1 was co-deleted in the Pten/p53 model, the lethality remained unperturbed. At the protein level, Chd1 was stabilized upon Pten deletion in prostate, but not in other organs examined (lung, liver, kidney, colon, mammary). These results shed mechanistic insight on the cancer type-specific copy number alteration pattern of PTEN and CHD1.

CHD1L promotes EOC cell invasiveness and metastasis via the regulation of METAP2.

Chromodomain helicase DNA binding protein 1-like (CHD1L) gene has been proposed to play an oncogenic role in human hepatocellular carcinoma. Previously we reported that CHD1L overexpression is significantly associated with the metastasis proceeding of epithelial ovarian cancer (EOC), and may predict a poor prognosis in EOC patients. However, the potential oncogenic mechanisms by which CHD1L acts in EOC remain unclear. To elucidate the oncogenic function of CHD1L, we carried out a series of in vitro assays, with effects of CHD1L ectogenic overexpression and silencing being determined in EOC cell lines (HO8910, A2780 and ES2). Real-time PCR and Western blotting analyses were used to identify potential downstream targets of CHD1L in the process of EOC invasion and metastasis. In ovarian carcinoma HO8910 cell lines, ectopic overexpression of CHD1L substantially induced the invasive and metastasis ability of the cancer cells in vitro. In contrast, knockdown of CHD1L using shRNA inhibited cell invasion in vitro in ovarian carcinoma A2780 and ES2 cell lines. We also demonstrated that methionyl aminopeptidase 2 (METAP2) was a downstream target of CHD1L in EOC, and we found a significant, positive correlation between the expression of CHD1L and METAP2 in EOC tissues (P<0.05). Our findings indicate that CHD1L plays a potential role in the inducement of EOC cancer cell invasion and/or metastasis via the regulation of METAP2 expression and suggests that CHD1L inhibition may provide a potential target for therapeutic intervention in human EOC.

Prostatic adenocarcinoma CNS parenchymal and dural metastases: alterations in ERG, CHD1 and MAP3K7 expression.

BACKGROUND: Prostatic carcinoma metastatic to dura is commonly encountered at autopsy, but presenting as a dural or, especially parenchymal, brain metastasis during life is far less common. Our group has been interested in two immunohistochemical (IHC) markers previously shown to be downregulated in particularly aggressive primary prostatic carcinomas: CHD1 and MAP3K7. Here we assess protein expression in clinically-relevant CNS metastases. We also assessed how these two markers correlated with the most common genetic alteration in prostate cancer: TMPRSS2 fusion to ERG (40-60% of carcinomas at the primary site), which places ERG expression under the control of the androgen-regulated TMPRSS2 gene, increasing expression. DESIGN: Database query, 2000-2016, identified 16 metastases to dura, 5 to brain parenchyma. RESULTS: Four of five intraparenchymal metastases and 15/16 informative dural-based metastases were ERG-negative (90.5% overall). There was reduced expression of CHD1 in 8/21 and reduced MAP3K7 in 17/21 cases; 7/19 (37%) ERG-negative metastases had dual low expression of CHD1/MAP3K7. ERG-positive cases had high expression of one or both markers. CONCLUSION: Metastatic prostatic carcinoma to CNS demonstrates expression patterns consistent with particularly aggressive behavior. Lower ERG expression in dural and intraparenchymal metastases suggests a possibility that ERG-negative tumors with loss of MAP3K7 may become resistant to standard therapies and diffusely metastasize.

Chd1p recognizes H3K36Ac to maintain nucleosome positioning near the transcription start site.

In Saccharomyces cerevisiae, the ATP-dependent chromatin remodeler, Chd1p, globally affects nucleosome positioning at coding regions, where nucleosomes are specifically and directionally aligned with respect to the transcription start site (TSS). Various auxiliary domains of remodelers play critical roles by performing specialized functions that are unique to the type of remodeler. Here, we report that yeast Chd1p directly binds to acetylated histone H3K36 (H3K36Ac) via its chromodomain, and that H3K36Ac stimulates the nucleosome sliding activity of Chd1p in vitro. Furthermore, we use genome-wide analysis to demonstrate that H3K36Ac promotes the remodeling activity of Chd1p to maintain chromatin stability at the 5' ends of genes in vivo. Our work linking Chd1p with H3K36Ac provides novel insights into how the nucleosome remodeling activity of Chd1p is controlled near the TSS.

CHD1 acts via the Hmgpi pathway to regulate mouse early embryogenesis.

The protein CHD1 is a member of the family of ATPase-dependent chromatin remodeling factors. CHD1, which recognizes trimethylated histone H3 lysine 4, has been implicated in transcriptional activation in organisms ranging from yeast to humans. It is required for pre-mRNA maturation, maintenance of mouse embryonic stem cell pluripotency and rapid growth of the mouse epiblast. However, the function(s) of CHD1 in mouse preimplantation embryos has not yet been examined. Here, we show that loss of CHD1 function led to embryonic lethality after implantation. In mouse embryos in which Chd1 was targeted by siRNA microinjection, the expression of the key regulators of cell fate specification Pou5f1 (also known as Oct4), Nanog and Cdx2 was dramatically decreased, starting at mid-preimplantation gene activation (MGA). Moreover, expression of Hmgpi and Klf5, which regulate Pou5f1, Nanog and Cdx2, was also significantly suppressed at zygotic gene activation (ZGA). Suppression of Hmgpi expression in Chd1-knockdown embryos continued until the blastocyst stage, whereas suppression of Klf5 expression was relieved by the morula stage. Next, we rescued HMGPI expression via Hmgpi mRNA microinjection in Chd1-knockdown embryos. Consequently, Pou5f1, Nanog and Cdx2 expression was restored at MGA and live offspring were recovered. These findings indicate that CHD1 plays important roles in mouse early embryogenesis via activation of Hmgpi at ZGA.